Emulsion based drug screening

ABSTRACT

The invention provides methods and systems for drug screening by segregating single cells into droplets simultaneously and providing candidate compound to the single cells to measure cellular response. Methods of the present invention combine template particles with a plurality of single cells in a tube, generate in the tube monodispersed droplets simultaneously that encapsulate a single one of the template particles and single one of the single cells, provide to the single cells one or more candidate compounds, and measure a cellular response to the one or more candidate compounds.

FIELD OF THE INVENTION

The invention relates to methods and systems for emulsion based drug screening.

BACKGROUND

Although recent advances in drug therapies have improved patient outcomes in some cancers, due to the complexity and heterogeneity of cancer cells there is no guarantee that any particular drug therapy will successfully result in remission and control of a patient's cancer. Moreover, remission and control can be fleeting, with drug targets changing as cancer cells continue to mutate and develop resistances to previously effective therapies.

Finding new drug therapies that may be effective against cancer requires extensive screening to pick out promising drug candidates against a vast number of available compounds. This is further exacerbated by the heterogeneity of cancer cells, which requires that each candidate compound be screened against any number of cell lines and cell types.

Numerous solutions to this problem have been proposed. For example, single-cell analysis has been proposed as a way to identify of potential drug therapies by providing scientists with the ability to segregate individual cell lines and cross them against candidate compounds. Traditional methods for segregating single cells employ flow cytometry and droplet microfluidics to separate single cells into individual droplets containing a drug compound one at a time. Those methods, however, require complicated equipment that is both expensive and difficult to use. Moreover, because each cell must be processed individually, such methods are rate limited and require extensive periods of time (often days) to separate and cross cells with candidate drugs. As a result, the cost of screening potential cancer therapies, both in time and money, have led to extended periods during which patients have endured treatments that are not effective as intended.

SUMMARY OF THE INVENTION

The invention provides methods and systems for drug screening that greatly reduce the cost, both in time and money, for screening cancer cells against candidate compounds. Methods of the present invention separate single cells in a sample simultaneously, rather than one by one, by encapsulating each cell into individual monodispersed droplets together with one or more candidate compounds. Because each droplet encapsulates only a single cell, cancer cell lines can be separated from one another and screened against candidate compounds. By separating cells into droplets simultaneously, rather than individually, these methods allow for the separation of any number of cancer cells from a sample within hours, rather than days. This provides for a rapid approach to generating a cells allowing for the testing of a greater number of candidate compounds at once. Additionally, methods of the present invention are performed without the need for complex and expensive machinery as required by microfluidic cell separation techniques, dramatically reducing the costs of screening potential therapies.

Moreover, methods of the present invention provide an approach that is easily scalable from small to large volumes of samples and can be automated. By reducing the complexity of single cell analysis, methods and systems of the present invention allow clinicians themselves to prepare samples from a patient for drug screening, increasing the availability of personalized drug monitoring and new therapy identification.

The present invention is achieved, in part, by combining template particles with a plurality of single cells in a tube, generating in the tube monodispersed droplets simultaneously that encapsulate a single one of the template particles and single one of the single cells, providing to the single cells one or more candidate compounds, and measuring a cellular response to the one or more candidate compounds.

Advantageously, each monodispersed droplet may be used to provide the one or more candidate compounds to the single cells. For example, the one or more candidate compounds may be provided to the monodispersed droplets by the single template particle encapsulated by each droplet. Because each droplet encapsulates only a single template particle, the template particles can be used to deliver a precise dose of the one or more candidate compounds.

Cellular response may be measured by any known methods. For example, methods for screening drug compounds may further comprise releasing nucleic acid molecules from the single cells and sequencing the nucleic acid molecules. This may comprise lysing each of the single cells contained within the monodisperse droplets to release the nucleic acid molecules. Once released, nucleic acid molecules may be sequenced, and the sequencing information may be used to measure a cellular response. For example, mRNA molecules may be used to measure gene expression levels associated with a drug response. This may be done by quantifying a plurality of distinct mRNA molecules in the cells associated with the drug response and using this information to generate a gene expression profile for each of the single cells. Cellular response may also be measured by quantifying a plurality of protein molecules in the cells associated with a drug response or be evaluating cellular pathways within each single cell. Because each cell may be analyzed according to cell line, and each cell within a cell line may be provided with different drug compounds, each candidate compound can be screened against multiple cell lines in order to generate a comprehensive drug screen.

Releasing nucleic acid molecules or proteins from single cells may comprise lysis of the single cells within the monodispersed droplets 10. Lysis may be induced by a stimulus such as heat, osmotic pressure, lytic reagents (e.g., DTT, beta-mercaptoethanol), detergents (e.g., SDS, Triton X-100, Tween-20), enzymes (e.g., proteinase K), or combinations thereof.

Template particles may provide candidate compounds to droplets through a variety of methods. For example, the one or more candidate compounds may be provided to the droplet from the surface of the template particle. Candidate compounds may be tethered to the surface of the template particle through the use of a linker, such as an oligonucleotide, covalent bond, or linker compound, or may be electromagnetically fixed to the surface of the template particle. The template particles may also comprise one or more compartments, each containing one or more candidate compounds that may be provided to the cells from a compartment of the template particle. Advantageously, an external stimulus, such as heat, may be used to release one or more candidate compounds from the one or more compartments to the cells. This allows for cells to be incubated with candidate compounds over a precise period of time.

The one or more candidate compounds may also be provided to the single cells after generating the plurality of monodispersed droplets. Accordingly, droplets may be designed such that candidate compounds may enter the monodispersed droplets after formation. Alternatively, providing the one or more candidate compounds to the single cells may also comprise the step of releasing the single cells from the monodispersed droplets prior to providing to the single cells one or more candidate drug compounds. Once released, the cells may be separate and then

crossed against the one or more candidate compounds, for example by using a microwell plate. Methods of the present invention simultaneously separate single cells by combining the template particles with the single cells in a first fluid, adding a second fluid to the first fluid, and shearing the fluids to generate a plurality of monodispersed droplets simultaneously that contain a single one of the template particles and a single one of the single cells.

In such methods the first fluid and the second fluid may be immiscible. For example, the first fluid may comprise an aqueous phase fluid and/or the second fluid may comprise an oil. The first fluid may comprise reagents selected from, for example, buffers, salts, lytic enzymes (e.g. proteinase k) and/or other lytic reagents (e. g. Triton X-100, Tween-20, IGEPAL, or combinations thereof), nucleic acid synthesis reagents e.g. nucleic acid amplification reagents or reverse transcription mix, or combinations thereof. The second fluid may comprise fluorocarbon oil, a silicone oil, or a hydrocarbon oil, or a combination thereof. Shearing fluids may comprise vortexing, shaking, flicking, stirring, pipetting, or any known method for mixing solutions.

Droplets generated by methods of the present invention are monodisperse and encapsulate a single one of the template particles and a single one of the single cells. Advantageously, the template particles may each provide one or more candidate compounds to the single cells. Because each droplet only encompasses one template particle and one single cell, by doing so the template particles provide a precise and uniform dose of each candidate compound to the single cells across droplets.

Template particles may comprise any known particles that can be used for forming the monodispersed droplets and advantageously may provide candidate compounds to each droplet. The template particles may be hydrogels, for example, hydrogels comprising agarose, alginate, a polyethylene glycol (PEG), a polyacrylamide (PAA), acrylate, acrylamide/bis-acrylamide copolymer matrix, azide-modified PEG, poly-lysine, polyethyleneimine, and combinations thereof. In certain instances, template particles may be shaped to provide an enhanced affinity for the single cells. For example, the template particles may be generally spherical but the shape may contain features such as flat surfaces, craters, grooves, protrusions, and other irregularities in the spherical shape that promote an association with a single cell such that the shape of the template particle increases the probability of templating a monodisperse droplet that contains a single cell.

Additionally, the template particles may further comprise one or more compartments. For example, the one or more compartments may contain one or more of a lytic reagent, a nucleic acid synthesis reagent, one or more candidate drug compounds, barcodes unique to each droplet, unique molecular identifiers (UMIs), or a combination thereof. It may be advantageous for the nucleic acid synthesis reagent to comprise a polymerase, for example when PCR is desired.

Tubes for single cell analysis of the present invention may be selected based on the volume of sample from which cells need to be separated and/or based on the number of cells to be separated. For example, the tube may be single large tube, such as a conical centrifuge tube, such as a Falcon® as sold by Corning Inc., Corning, N.Y., for example a tube with a volume of less than 40 mL. The tubes may also be wells, such as standard 96 sample well kits. The tubes may also be centrifuge, microcentrifuge, or PCR tubes, such as those sold be Eppendorf ®, Hamburg, Germany. Such tubes, for example, may be between 0.1 and 6 mL.

For any tubes sample preparation for sequencing may be completed within one day, and advantageously can be completed within three hours. Moreover, preparation of samples within each tube may be completed in as little time as about 5 minutes or about 2 minutes. This is in contrast to preparation of cells by microfluidics which often require three days for sample preparation, and further advantageous over prior emulsion based preparations which required, at least, additional steps and time for providing candidate compounds to each cell.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a monodispersed droplet according to one aspect of the invention.

FIG. 2 shows a micrograph of a monodispersed droplet containing a flat faceted template particle according to an embodiment of FIG. 1 .

FIG. 3 shows a schematic representation of a monodispersed droplet containing an internal compartment.

FIG. 4 shows a schematic representation of a monodispersed droplet containing an internal compartment.

FIG. 5 shows a schematic representation of a monodispersed droplet after release of nucleic acid molecules or proteins from a single cell.

FIG. 6 shows a schematic representation of a method for single cell analysis according to some aspects of the present disclosure.

FIG. 7 shows a schematic representation of a monodispersed droplet after release of nucleic acid molecules or proteins from a single cell.

FIG. 8 shows a schematic representation of a monodispersed droplet with capture probes.

FIG. 9 diagrams a method for single cell analysis according to other aspects of the present disclosure.

FIG. 10 shows a schematic representation of a monodispersed droplet after release of nucleic acid molecules or proteins from a single cell and dissolution of the template particle.

FIG. 11 shows a schematic representation of a monodispersed droplet with capture probes.

FIG. 12 shows a representation of a capture probe.

FIG. 13 shows a representation of first complimentary strand synthesis.

FIG. 14 shows a representation of second complimentary strand synthesis

FIG. 15 shows a schematic representation of monodispersed droplets in a tube.

FIG. 16 shows a schematic representation of a method for rupturing monodispersed droplets.

DETAILED DESCRIPTION

The present invention provides methods and systems of analyzing single cells by combining template particles with a plurality of single cells in a tube, generating in the tube monodispersed droplets simultaneously that encapsulate a single one of the template particles and single one of the single cells, providing to the single cells one or more candidate compounds, and measuring a cellular response to the one or more candidate compounds. Cellular response to the one or more candidate compounds may be measured by any known method, for example from a gene expression profile, protein expression profile, or cellular signaling analysis.

Template particles may provide candidate compounds to droplets. Template particles may provide candidate compounds to droplets through a variety of methods. For example, the one or more candidate compounds may be provided to the droplet from the surface of the template particle. Candidate compounds may be tethered to the surface of the template particle through the use of a linker, such as an oligonucleotide, covalent bond, or linker compound, or may be electromagnetically fixed to the surface of the template particle. The template particles may also comprise one or more compartments, each containing one or more candidate compounds that may be provided to the cells from a compartment of the template particle. Advantageously, an external stimulus, such as heat, may be used to release one or more candidate compounds from the one or more compartments to the cells. This allows for cells to be incubated with candidate compounds over a precise period of time.

The one or more candidate compounds may also be provided to the single cells after generating the plurality of monodispersed droplets. Accordingly, droplets may be designed such that candidate compounds may enter the monodispersed droplets after formation. Alternatively, providing the one or more candidate compounds to the single cells may also comprise the step of releasing the single cells from the monodispersed droplets prior to providing to the single cells one or more candidate drug compounds. Once released, the cells may be separate and then crossed against the one or more candidate compounds, for example by using a microwell plate.

Candidate compounds screened by methods of the present invention may be any potential drug compounds. Template particles, droplets, and methods for introducing candidate compounds to cells may be designed based on the candidate compounds to be screened. Candidate compounds, for example, may be selected from a group consisting of small molecules, biologics, antibodies, proteins, and polypeptides.

Cellular response may be measured by any known methods. For example, methods for screening drug compounds may further comprise releasing nucleic acid molecules from the single cells and sequencing the nucleic acid molecules. This may comprise lysing each of the single cells contained within the monodisperse droplets to release the nucleic acid molecules. Once released, nucleic acid molecules may be sequenced, and the sequencing information may be used to measure a cellular response. For example, mRNA molecules may be used to measure gene expression levels associated with a drug response. This may be done by quantifying a plurality of distinct mRNA molecules in the cells associated with the drug response and using this information to generate a gene expression profile for each of the single cells. Cellular response may also be measured by quantifying a plurality of protein molecules in the cells associated with a drug response or be evaluating cellular pathways within each single cell. Because each cell may be analyzed according to cell line, and each cell within a cell line may be provided with different drug compounds, each candidate compound can be screened against multiple cell lines in order to generate a comprehensive drug screen.

Advantageously, cellular response once measured may be compared against a reference cellular response. For example, a gene expression profile for a cell screened against a candidate compound may be compared against a cell provided a control compound, against the gene expression profile of a different patient, against a pooled gene expression profile from a number of different subjects, and/or against a gene expression previously measured in the patient.

Releasing nucleic acid molecules or proteins from single cells may comprise lysis of the single cells within the monodispersed droplets 10. Lysis may be induced by a stimulus such as heat, osmotic pressure, lytic reagents (e.g., DTT, beta-mercaptoethanol), detergents (e.g., SDS, Triton X-100, Tween-20), enzymes (e.g., proteinase K), or combinations thereof.

Template particles may provide candidate compounds to droplets through a variety of methods. For example, the one or more candidate compounds may be provided to the droplet from the surface of the template particle. Candidate compounds may be tethered to the surface of the template particle through the use of a linker, such as an oligonucleotide, covalent bond, or linker compound, or may be electromagnetically fixed to the surface of the template particle. The template particles may also comprise one or more compartments, each containing one or more candidate compounds that may be provided to the cells from a compartment of the template particle. Advantageously, an external stimulus, such as heat, may be used to release one or more candidate compounds from the one or more compartments to the cells. This allows for cells to be incubated with candidate compounds over a precise period of time.

Template particles may comprise any known particles that can be used for forming the monodispersed droplets and advantageously may provide one or more candidate drug compounds to each droplet. Template particles for single cell analysis leverage the particle-templated emulsification technology previously described in, Hatori et. al., Anal. Chem., 2018 (90):9813-9820, which is incorporated by reference. Most frequently, micron-scale beads (such as hydrogels) may be used to define an isolated fluid volume surrounded by an immiscible partitioning fluid and stabilized by temperature insensitive surfactants.

The template particles of the present disclosure may be prepared using any method known in the art. Generally, the template particles are prepared by combining hydrogel material, e.g., agarose, alginate, a polyethylene glycol (PEG), a polyacrylamide (PAA), Acrylate, Acrylamide/bis-acrylamide copolymer matrix, and combinations thereof. Following the formation of the template particles they are sized to the desired diameter for capturing and uniquely tagging cells. For example, sizing of the template particles may be done by microfluidic co-flow into an immiscible oil phase.

Template particles may vary in size. Variation may be limited, for example the diameter or largest dimension of the template particles may be such that at least 50% or more, e.g., 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, or 99% or more of the template particles vary in diameter or largest dimension by less than a factor of 10, e.g., less than a factor of 5, less than a factor of 4, less than a factor of 3, less than a factor of 2, less than a factor of 1.5, less than a factor of 1.4, less than a factor of 1.3, less than a factor of 1.2, less than a factor of 1.1, less than a factor of 1.05, or less than a factor of 1.01.

Advantageously, the absorbency of the presently disclosed template particles may be increased by storing them in a dehydrated condition prior to using them in the presently disclosed method for single cell analysis, with the general intention of shrinking their volume. Shrinking template particles allows for control of the template particle shape and size for capturing cells and for controlling the solution to candidate drug ratio in droplets. For example, dehydration of the template particles may be achieved by storing them in a high osmolarity buffer to promote shrinking (i.e. Polyethelene glycol). Alternatively, the template particles may be may be ethanol dehydrated. Shrinking may occur upon the application of an external stimulus, e.g., heat. For instance, advantageously the template particles may be encapsulated in a fluid by shearing, followed by the application of heat, causing the template particles to shrink in size. Some other examples of drying approaches include, but are not limited to, heating, drying under vacuum, freeze drying, and supercritical drying. The dried template particles may also be combined with a fluid, but still retain the shape and structure as independent, often spherical, gel particles. The dried template particles may be combined with an appropriate fluid, causing a portion of the fluid to be absorbed by the template particles. Porosity of the template particles may also vary, to allow at least one of a plurality of cells to be absorbed into the template particles when combined with the appropriate fluid. Any convenient fluid that allows for the desired absorption to be performed in the template particles may be used.

Template particles are advantageously tiny, generally spherical, particles. Template particles may be porous or nonporous. Template particles may also include microcompartments or internal compartments which advantageously may contain additional components and/or reagents, for example, additional components and/or reagents that may be releasable into monodisperse droplets. Advantageously, template particles may include microcompartments which include the one or more candidate compounds in order to provide the candidate compounds to the cells in the droplet.

Template particles for such use may include a polymer such as a hydrogel. Template particles generally range from about 0.1 to about 1000 μm in diameter or largest dimension. Template particles may have a diameter or largest dimension of about 1.0 μm to 1000 μm, inclusive, such as 1.0 μm to 750 μm, 1.0 μm to 500 μm, 1.0 μm to 250 μm, 1.0 μm to 200 μm, 1.0 μm to 150 μm 1.0 μm to 100 μm, 1.0 μm to 10 μm, or 1.0 μm to 5 82 m, inclusive. Template particles may have a diameter or largest dimension of about 10 μm to about 200 82 m, e.g., about 10 μm to about 150 μm, about 10 μm to about 125 82 m, or about 10 μm to about 100 μm.

Cells analyzed by the present invention may include live cells obtained from, for example, a sample (tissue of bodily fluid) of a patient. The sample may include a fine needle aspirate, a biopsy, or a bodily fluid from the patient. Upon being isolated from the sample, the cells may be processed by, for example, generating a single cell suspension with an appropriate solution. Such solution will generally be a balanced salt solution, e.g. normal saline, PBS, HBSS (Hank's balanced salt solution), etc., and in certain instances supplemented with fetal calf serum or other naturally occurring factors, in conjunction with an acceptable buffer at low concentration, generally from 5-25 mM. Convenient buffers include HEPES, phosphate buffers, lactate buffers, etc. The separated cells can be collected in any appropriate medium that maintains the viability of the cells, usually having a cushion of serum at the bottom of the collection tube. Various media are commercially available and may be used according to the nature of the cells, including dMEM, HBSS, DPBS, RPMI, IMDM (Iscove's medium), etc., frequently supplemented with fetal calf serum.

Preferably, the cells are mammalian cells, for example human cells. A person of skill in the art will understand that cells can be selected based on the drug to be screened. For example, potential cancer therapies may be screened against cancer cells, for example from a tumor biopsy. Alternatively, candidate compounds for a liver disorder, such as non-alcoholic fatty liver disease (NAFLD) or non-alcoholic steatohepatitis (NASH), may be screened against liver cells, for example a hepatocyte. Methods of the present invention may be applied to any cell type or cell line, providing for cells to be individually screened against candidate compounds.

In selecting a cell type for candidate compound screening, the composition and nature of the template particles may vary depending on the cell types against which candidate drugs are being screened. For instance, the template particles may be microgel particles that are micron-scale spheres of gel matrix. The microgels are composed of a hydrophilic polymer that is soluble in water, including alginate or agarose. Microgels may also be composed of a lipophilic microgel.

Template particles may also be a hydrogel, such as hydrogels from naturally derived materials, synthetically derived materials, and combinations thereof. Examples of hydrogels include, but are not limited to, collagen, hyaluronan, chitosan, fibrin, gelatin, alginate, agarose, chondroitin sulfate, polyacrylamide, polyethylene glycol (PEG), polyvinyl alcohol (PVA), acrylamide/bis-acrylamide copolymer matrix, polyacrylamide/poly(acrylic acid) (PAA), hydroxyethyl methacrylate (HEMA), poly N-isopropylacrylamide (NIPAM), and polyanhydrides, poly(propylene fumarate) (PPF).

Template particles may further advantageously comprise materials which provide the template particles with a positive surface charge, or an increased positive surface charge. Such materials may be without limitation poly-lysine or Polyethyleneimine, or combinations thereof. This may increase the chances of association between the template particle and, for example, a cell which generally have a mostly negatively charged membrane.

Other strategies aimed to increase the chances of template particle-cell association include creation of a specific template particle geometry. For example, the template particles may have a general spherical shape but the shape may contain features such as flat surfaces, craters, grooves, protrusions, and other irregularities in the spherical shape. The surface template geometry of template particles may also be used to tether and/or provide one or more candidate compounds to the droplets and thereby to the cells within the droplets. Template geometry may also be used to ensure that the single cell and candidate compounds are localized within the droplet to facilitate cell to candidate compound interaction.

Any one of the above described strategies and methods, or combinations thereof may be used in the practice of the presently disclosed template particles and method for single cell analysis thereof. Methods for generation of template particles, and template particles-based encapsulations, were described in International Patent Publication WO 2019/139650, which is incorporated herein by reference.

Creating template particle-based encapsulations for single cell analysis may comprise combining single cells with a plurality of template particles in a first fluid to provide a mixture in a reaction tube. The mixture may be incubated to allow association of the plurality of the template particles with single cells. A portion of the plurality of template particles may become associated with the single cells. The mixture is then combined with a second fluid which is immiscible with the first fluid. The fluid and the mixture are then sheared so that a plurality of monodispersed droplets is generated within the reaction tube. The monodisperse droplets generated comprise (i) at least a portion of the mixture, (ii) a single template particle, and (iii) a single cell. Advantageously, the template particles may provide on or more candidate compounds to the monodisperse droplets. Of note, in practicing methods of the invention provided by this disclosure a substantial number of the monodispersed droplets generated will comprise a single template particle and a single cell, however, in some instances, a portion of the monodispersed droplets may comprise none or more than one template particle or cell. In such instances, monodispersed droplets comprising no cells, greater than one cell, no template particles and/or or greater than one template particle (are therefore greater than one barcode) may be excluded from further analysis. In the case of droplets with greater than one template or greater than one cell, it is advantageous to exclude these droplets because each template particle may be designed to deliver a precise dosage of a candidate compound to each cell.

FIG. 1 shows a monodispersed droplet according to one aspect of the invention. The depicted monodispersed droplet 10 comprises a template particle 1, a single cell 3. The template particle illustrated comprises flat facets 2, and provides a candidate compound 11 to the droplet. FIG. 2 shows a micrograph of flat faceted template particles 1 according to an embodiment of FIG. 1 . Each monodispersed droplet 1 in FIG. 2 contains one or more candidate compounds 11 to be crossed against the single cell within the droplet. In some embodiments, the first fluid is an aqueous phase fluid and the second fluid is an oil, e.g. fluorocarbon oil, a silicone oil, or a hydrocarbon oil, or a combination thereof.

To increase the chances of generating an encapsulation, such as, a monodispersed droplet 10 that contains one template particle 1 and one single cell 3, the template particles and cells may be combined at a ratio wherein there are more template particles than cells. For example, the ratio of template particles to cells combined in a mixture as described above may be in a range of 5:1 to 1,000:1, respectively. The template particles and cells may also be combined at a ratio of 10:1, 100:1, or 1000:1, respectively. Excess template particles may form monodisperse droplets without any cells within the droplet. As described above, these droplets may be removed from analysis.

To generate a monodisperse emulsion 10, a step of shearing the second mixture provided by combining a first mixture comprising template particles and cells with a second fluid immiscible with the first mixture. Any suitable method may apply a sufficient shear force to the second mixture. For example, the second mixture may be sheared by flowing the second mixture through a pipette tip. Other methods include, but are not limited to, shaking the second mixture with a homogenizer (e.g., vortexer), or shaking the second mixture with a bead beater. Vortexing may be performed for example for 30 seconds, or in the range of 30 seconds to 5 minutes. The application of a sufficient shear force breaks the second mixture into monodisperse droplets that encapsulate one of a plurality of template particles.

Generating the template particles-based monodisperse droplets may involve shearing two liquid phases. For example, the mixture may be the aqueous phase and comprise reagents selected from, for example, buffers, salts, lytic enzymes (e.g. proteinase k) and/or other lytic reagents (e.g. Triton X-100, Tween-20, IGEPAL, bm 135, or combinations thereof), nucleic acid synthesis reagents e.g. nucleic acid amplification reagents, or combinations thereof. The fluid may be the continuous phase and may be an immiscible oil such as fluorocarbon oil, a silicone oil, or a hydrocarbon oil, or a combination thereof. The fluid may advantageously comprise reagents such as surfactants (e.g. octylphenol ethoxylate and/or octylphenoxypolyethoxyethanol), reducing agents (e.g. DTT, beta mercaptoethanol, or combinations thereof).

In practicing methods as described herein, the composition and nature of the monodisperse droplets, e.g., single-emulsion and multiple-emulsion droplets, may vary. Advantageously, a surfactant may be used to stabilize the droplets 10. The monodisperse droplets described herein may be prepared as emulsions, e.g., as an aqueous phase fluid dispersed phase carrier fluid (e.g., a fluorocarbon oil, silicone oil, or a hydrocarbon oil) or vice versa. Accordingly, a droplet may involve a surfactant stabilized emulsion, e.g., a surfactant in an immiscible stabilized single emulsion or a surfactant stabilized double emulsion. Any convenient surfactant that allows for the desired reactions to be performed in the droplets may be used. In other aspects, monodisperse droplets are not stabilized by surfactants.

FIG. 3 is a schematic representation of a single monodispersed droplet according to another embodiment of the present invention. The depicted monodispersed droplet 10 comprises a template particle 1 and a single cell 3. The template particle 3 illustrated comprises crater-like depressions 2, and, in the embodiment illustrated, the single cell 3 is associated with one of the crater-like depressions 2. The template particle 3 may comprise further crater like depressions 2 associated with one or more candidate compounds 11. The single cell 3 further comprises at least one internal compartment 4. The internal compartment 4 may alternatively or additionally contain the one or more candidate compounds 11.

As described above, the template particles may contain multiple internal compartments 4. The internal compartments 4 of the template particles 1 may also be used to provide reagents that can be triggered to release a desired compound, e.g., a substrate for an enzymatic reaction, or induce a certain result, e.g. lysis of an associated single cell 3. Reagents provided in the template particles' compartment 4 may be without limitation reagents selected from buffers, salts, lytic enzymes (e.g. proteinase k), other lytic reagents (e. g. Triton X-100, Tween-20, IGEPAL, bm 135), nucleic acid synthesis reagents, or combinations thereof.

The internal compartment 4 may also be used to provide any additional components, for example component used for measuring a cellular response to a candidate compound. Additional components provided by the internal compartment may comprise components used for processing proteins or nucleic acid molecules, for example for nucleic acid amplification, reverse transcription, sequencing, or gene expression. Components used for sequencing nucleic acids may include barcodes unique to each template particle 1, and therefore to the droplet 10. When nucleic acid molecules or proteins are released from the cell 3, they may be tagged with the droplet specific barcode provided by the template particle 1. Each nucleic acid molecule or protein can then be identified with the source template particle 1, droplet 10, and cell 3.

The barcodes may be any group of nucleotides or oligonucleotide sequences that are distinguishable from other barcodes within the group. A droplet encapsulating a template particle and a single cell provides to each nucleic acid molecule or protein released from the single cell the same barcode from the group of barcodes. The barcodes provided by each droplet are unique to that droplet and distinguishable from the barcodes provided to nucleic acid molecule or protein by every other droplet. Once sequenced, by using the barcode sequence, the nucleic acid molecules or proteins can be traced back to the droplet and thereby to each single cell. Barcodes may be of any suitable length sufficient to distinguish the barcode from other barcodes. For example, a barcode may have a length of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 nucleotides, or more. The barcodes may be pre-defined, degenerate, and/or selected at random.

Barcodes may be added to nucleic acid molecules or proteins by “tagging” the nucleic acid molecules with the barcode. Tagging may be performed using any known method for barcode addition, for example direct ligation of barcodes to one or more of the ends of each nucleic acid molecule. Nucleic acid molecules may, for example, be end repaired in order to allow for direct or blunt-ended ligation of the barcodes. Barcodes may also be added to nucleic acid molecules through first or second strand synthesis, for example using capture probes, as described herein below.

The internal compartment may also provide unique molecule identifiers (UMIs) to nucleic acids. Unique molecule identifiers (UMIs) are a type of barcode that may be provided to nucleic acid molecules in a sample to make each nucleic acid molecule, together with its barcode, unique, or nearly unique. This is accomplished by adding, e.g. by ligation, one or more UMIs to the end or ends of each nucleic acid molecule such that it is unlikely that any two previously identical nucleic acid molecules, together with their UMIs, have the same sequence. By selecting an appropriate number of UMIs, every nucleic acid molecule in the sample, together with its UMI, will be unique or nearly unique. One strategy for doing so is to provide to a sample of nucleic acid molecules a number of UMIs in excess of the number of starting nucleic acid molecules in the sample. By doing so, each starting nucleic molecule will be provided with different UMIs, therefore making each molecule together with its UMIs unique. However, the number of UMIs provided may be as few as the number of identical nucleic acid molecules in the original sample. For example, where no more than six nucleic acid molecules in a sample are likely to be identical, as few as six different UMIs may be provided, regardless of the number of starting nucleic acid molecules.

UMIs are advantageous in that they can be used to correct for errors created during amplification, such as amplification bias or incorrect base pairing during amplification. For example, when using UMIs, because every nucleic acid molecule in a sample together with its UMI or UMIs is unique or nearly unique, after amplification and sequencing, molecules with identical sequences may be considered to refer to the same starting nucleic acid molecule, thereby reducing amplification bias. Methods for error correction using UMIs are described in Karlsson et al., 2016, Counting Molecules in cell-free DNA and single cells RNA”, Karolinska Institutet, Stockholm Sweden, available at <https://openarchive.ki.se/xmlui/handle/10616/45053>, incorporated herein by reference.

FIG. 4 shows a schematic representation of another embodiment of one of a plurality of monodispersed droplets 10. The depicted monodispersed droplet 10 in FIG. 4 comprises a template particle 1 and a single cell 3. The template particle 1 illustrated is generally spherical, and in the embodiment illustrated, the single cell 3 is associated with the template particle 1. The template particle 1 further comprises an internal compartment 4, the internal compartment 4 comprises reagents. A second internal compartment provides candidate drug compound 11.

FIG. 5 shows a schematic representation of the monodispersed droplet following an external stimulus 6. After the external stimulus 6 is applied, reagents are activated and released, dissolving 8 the template particle 1, while the monodispersed droplet 10 remains intact as depicted by the intact encapsulation shell 5. In some embodiments, the external stimulus 6 may be heat or osmotic pressure. Within the droplet, the drug compound is released from the internal compartment 4 of the template particle 5 and may be screened against the single cell 3.

As depicted in FIG. 3 , one or more of the said reagents (e.g., detergents, enzymes) may be compartmentalized 4 within the template particle 1. In other embodiments, one or more of the said reagents is present in the mixture. In some other embodiments, one or more of the said reagents is added to the solution comprising the monodisperse droplets 10, as desired.

Methods of the invention comprise measuring cellular response to one or more candidate compounds. Methods may include releasing nucleic acid molecules from single cells 3 segregated inside monodispersed droplets 10, tagging each nucleic acid molecule with barcode unique to the monodispersed droplet, and then sequencing the nucleic acid molecule. Sequencing may analyze genomic areas of interest, e.g. oncogenes. Thus, PCR amplification of products derived from nucleic acid molecules released by single cells can be used to determine a cellular response to a candidate compound. For example, quantification of mRNA can provide gene expression profile, a measurement of cellular response, for a gene of interest, for example a gene associated with cancer. Genes and mutations of interest may include, but are not limited to, BAX, BCL2L1, CASP8, CDK4, ELK1, ETS1, HGF, JAK2, JUNB, JUND, KIT, KITLG, MCL1, MET, MOS, MYB, NFKBIA, EGFR, Myc, EpCAM, NRAS, PIK3CA, PML, PRKCA, RAF1, RARA, REL, ROS1, RUNX1, SRC, STAT3, CD45, cytokeratins, CEA, CD133, HER2, CD44, CD49f, CD146, MUC1/2, ABL1, AKT1, APC, ATM, BRAF, CDH1, CDKN2A, CTNNB1, EGFR, ERBB2, ERBB4, EZH2, FBXW7, FGFR2, FGFR3, FLT3, GNAS, GNAQ, GNA11, HNF1A, HRAS, IDH1, IDH2, JAK2, JAK3, KDR, KIT, KRAS, MET, MLH1, NOTCH1, NPM1, NRAS, PDGFRA, PIK3CA, PTEN, PTPN11, RB1, RET, SMAD4, STK11, TP53, VHL, and ZHX2. Measuring cellular response may also comprise measuring expression levels of a protein associated with drug response.

For example, quantification of mRNA expressing the sequence of a gene or mutation of interest may provide information that the cell from which the nucleic acid molecule was released has increased expression of mRNA associated with cancer or decreased expression of mRNA associated with cancer in response to a candidate compound. Because each nucleic acid molecule may be tagged with a barcode unique to the droplet and single cell from which it was released, gene expression levels may be traced back to the droplet, template particle, and cell from which the mRNA was released. This allows for determination of the cell type and cell line expressing the mRNA and the one or more candidate drugs provided to the cell. Because methods of the present invention allow any number of monodisperse droplets to be generated simultaneously, any number of cell types and cell lines may be screened against any number of candidate compounds simultaneously. By using barcodes and UMIs gene expression levels can be traced back to each monodispersed droplet, allowing for minimal to no loss of information.

For RNA or mRNA sequencing, sequencing may first comprise the step of preparing a cDNA library from barcoded RNA, through reverse transcription, and sequencing the cDNA. RNA sequencing may advantageously allow for the quantification of gene expression within the single cell, and can be used to identify characteristics of the single cell that can be used to, for example, make a diagnosis, prognosis, or determine drug effectiveness. Reverse transcription of cDNA molecules from RNA can be performed both within the droplet or after barcoded RNA molecules have been released from each droplet.

Reverse transcription may be performed using without limitation dNTPs (mix of the nucleotides dATP, dCTP, dGTP and dTTP), buffer/s, detergent/s, or solvent/s, as required, and suitable enzyme such as polymerase or reverse transcriptase. The polymerase used may be a DNA polymerase, and may be selected from Taq DNA polymerase, Phusion polymerase (as provided by Thermo Fisher Scientific, Waltham, Mass.), or Q5 polymerase. Nucleic acid amplification reagents are commercially available, and may be purchased from, for example, New England Biolabs, Ipswich, Mass., USA. The reverse transcriptase used in the presently disclosed targeted library preparation method may be for example, maxima reverse transcriptase. In some embodiments, the general parameters of the reverse transcription reaction comprise an incubation of about 15 minutes at 25 degrees and a subsequent incubation of about 90 minutes at 52 degrees.

Sequencing nucleic acid molecules may be performed by methods known in the art. For example, see, generally, Quail, et al., 2012, A tale of three next generation sequencing platforms: comparison of Ion Torrent, Pacific Biosciences and Illumina MiSeq sequencers, BMC Genomics 13:341. Nucleic acid molecule sequencing techniques include classic dideoxy sequencing reactions (Sanger method) using labeled terminators or primers and gel separation in slab or capillary, or preferably, next generation sequencing methods. For example, sequencing may be performed according to technologies described in U.S. Pub. 2011/0009278, U.S. Pub. 2007/0114362, U.S. Pub. 2006/0024681, U.S. Pub. 2006/0292611, U.S. Pat. Nos. 7,960,120, 7,835,871, 7,232,656, 7,598,035, 6,306,597, 6,210,891, 6,828,100, 6,833,246, and 6,911,345, each incorporated by reference.

The conventional pipeline for processing sequencing data includes generating FASTQ-format files that contain reads sequenced from a next generation sequencing platform, aligning these reads to an annotated reference genome, and quantifying expression of genes. These steps are routinely performed using known computer algorithms, which a person skilled in the art will recognize can be used for executing steps of the present invention. For example, see Kukurba, Cold Spring Harb Protoc, 2015 (11):951-969, incorporated by reference.

The invention provides methods for screening cancer at early stages when the tumor size is smallest and the number of cancer cells is limited. The method includes isolating a plurality of single cells from the heterogeneous cell population by combining the heterogeneous cells with a plurality of template particles in a first fluid, adding a second fluid that is immiscible with the first fluid, and shearing the fluids to generate an emulsion comprising monodispersed droplets that each contain a single cell and a single template particle. Because cells are individually separated within monodisperse droplets, candidate compounds may be provided to each cell individually. Accordingly, when as few as 10,000 cancer cells from a cancer cell line are obtained from a patient, cells can be dividing into 1,000 groups of 10 cells each, each group screening a candidate compound. The method allows for drug screening in both low and high volumes of sample, wherein each cell is simultaneously separated.

For example, the method allows for analysis of a heterogeneous tumor biopsy taken from a subject. The method includes obtaining a biopsy from a patient and isolating a population of cells from the biopsy. The method further includes segregating the population of cells taken from the biopsy into droplets by combining the population of cells with a plurality of template particles in a first fluid, adding a second fluid that is immiscible with the first fluid, and shearing the fluids to generate an emulsion comprising monodispersed droplets that each contain a single one of the population of cells and a single template particle. Each cell is provided with a candidate compound to be screened and evaluated for cellular response to the candidate compound.

Nucleic Acid molecules may advantageously be amplified prior to sequencing. Amplification may comprise methods for creating copies of nucleic acids by using thermal cycling to expose reactants to repeated cycles of heating and cooling, and to permit different temperature-dependent reactions (e.g. by Polymerase chain reaction (PCR). Any suitable PCR method known in the art may be used in connection with the presently described methods. Non limiting examples of PCR reactions include real-time PCR, nested PCR, multiplex PCR, quantitative PCR or touchdown PCR. Notably, each amplified copy of the nucleic acid molecule will comprise the barcode unique to a droplet for identifying the droplet and cell form which the nucleic acid molecule was released.

Template particles may also comprise a plurality of capture probes. Generally, a capture probe is an oligonucleotide. The capture probes may attach to the template particle's material via covalent acrylic linkages. The capture probes may comprise an acrydite-modified on their 5′ end (linker region). Generally, acrydite-modified oligonucleotides can be incorporated, stoichiometrically, into hydrogels such as polyacrylamide, using standard free radical polymerization chemistry, where the double bond in the acrydite group reacts with other activated double bond containing compounds such as acrylamide. Specifically, copolymerization of the acrydite-modified capture probes with acrylamide including a crosslinker, e.g. N,N′-Methylenebis, will result in a crosslinked gel material comprising covalently attached capture probes. Capture probes may also comprise Acrylate terminated hydrocarbon linker and combining the said capture probes with a template particle will cause their attachment to the template particle.

The capture probe may comprise one or more of a primer sequence, the barcode unique to each droplet, a unique molecule identifier (UMI), and/or a capture sequence.

Primer sequences may comprise a binding site, for example a primer sequence that would be expected to hybridize to a complementary sequence, if present, on any nucleic acid molecule or protein released from a cell and provide an initiation site for a reaction, for example an elongation or polymerization reaction. The primer sequence may also be a “universal” primer sequence, i.e. a sequence that is complimentary to nucleotide sequences that are very common for a particular set of nucleic acid fragments. The primer sequences used may be P5 and P7 primers as provided by Illumin, Inc., San Diego, Calif. The primer sequence may also allow the capture probe to bind to a solid support, such as a template particle.

By providing capture probes comprising the barcode unique to each droplet, the capture probes may be use to tag the nucleic molecules or proteins released from single cells with the barcode. This process, discussed further herein below, may comprise hybridizing the nucleic acid molecule to the capture probe followed by an amplification or reverse transcription reaction.

Capture sequences used in capture probes are advantageous for targeting specific nucleotide sequences or proteins, for example nucleotide sequences known to be associated with a particular cancer genotype or phenotype. In such methods, the target nucleic sequence, if present, attaches to the template particle by hybridizing to the capture sequence upon release from the single cells.

FIG. 6 show a schematic representation of monodispersed droplet. The depicted monodispersed droplet 10 comprises a template particle 1 and a single cell 3. The template particle 1 illustrated is generally spherical and comprises multiple capture probes 12, which may advantageously comprise the barcode unique to the droplet. Reagents, such as lytic reagents 11, are present within the monodispersed droplet 10. The cell 3 has been provided with a candidate compound 11.

FIG. 7 shows the monodispersed droplet of FIG. 6 following an external stimulus 6. The cell 3 has been provided with the candidate compound 11 for screening. Following the stimulus 6 lytic reagents 11 are activated, lysing the single cell 3, while the encapsulation, i.e., monodispersed droplet 10 (depicted intact encapsulation shell 5) and template particle 1 remain intact. Upon lysis of the single cell 3, nucleic acid molecules and proteins 15 previously contained in the cell are released into the monodispersed droplet 10. A portion of the released nucleic acid molecules or proteins 15 associate with a portion of the capture probes 12 as depicted in FIG. 8 . Advantageously, the capture probe may comprise the barcode unique to the monodispersed droplet and may be used to tag the nucleic acid molecule or protein with the barcode.

Nucleic acid molecules or proteins hybridized to capture probes may be released for example by dissolving the template particle or by using a reducing reagent to reduce the dithiol oligonucleotide modifications in a capture probe linker region. Steps subsequent to the association of nucleic acid molecules or proteins and capture probes, such as amplification or nucleic acid molecules, may be done either inside the encapsulations or in bulk. When performing steps in bulk, an aqueous solution comprising the medium inside the encapsulations is generated upon breakage of the encapsulations. Any reagents, such as lytic reagents or nucleic acid synthesis reagents may be supplied in bulk, provided upon creation of the partitions (e.g., present in the first mixture), compartmentalized within the template particles, or combinations thereof.

FIG. 9 shows a schematic representation a method for single cell analysis according to other aspects of the present disclosure. Depicted is a single monodispersed droplet 10 from among a plurality of monodispersed droplets, which comprises a template particle 3 and a target cell 1. The template particle 1 comprises multiple capture probes 12. As described above, the each capture probe may comprise the barcode unique to the droplet. Reagents, such as lytic reagents 11, are present within the monodispersed droplet 10. The cell 3 has been provided with a candidate compound 11.

FIG. 10 shows the monodispersed droplet of FIG. 9 following an external stimulus 6. The cell 3 has been provided with the candidate compound 11 for screening. Following an external stimulus 6 the lytic reagents 11 are activated, lysing the single cell 3 and dissolving the template particle 1, however the monodispersed droplet 10 remains intact (depicted is the encapsulation shell 5). As the template particle 1 dissolves the capture probes 12 are released from it. Upon lysis of the single cell 3, nucleic acid molecules and proteins 15 previously contained in the single cell 3 are released. A portion of the released nucleic acid molecules 15 associates with the capture probes 12, as depicted in FIG. 11 . Even without the template particle present, the capture probe may comprise the barcode unique to the monodispersed droplet and may be used to tag the nucleic acid molecule or proteins with the barcode. Cellular response to the candidate compound 11 may than be measured.

FIG. 12 shows a method of barcoding a nucleic acid molecules released from a cell that has been provided with a candidate compound. As illustrated, the template particle 1 comprises a plurality of capture probes 12 illustrated schematically by curved broken lines. One of the capture probes 12 is featured in a larger scale and in detail. The capture probe 12 preferably comprises, from 5′ end to 3′ end, a linker region to allow covalent bond with the template particle 1, a “PR1” nucleotide sequence region comprising a primer nucleotide sequence, at least one UMI, a barcode unique to the droplet 201 (“BRCD”), and a capture nucleotide sequence 22 comprising a sequence complimentary to the nucleic acid molecule.

FIG. 13 shows a released nucleic acid molecule 15 comprising, optionally, a tail sequence. The nucleic acid molecule attaches to the capture probe of FIG. 12 's complimentary sequence 22 via complementary base pairing. For RNA molecules, the poly-A tail of an RNA molecule may be used to attach the RNA molecule to the capture probe, for example by using a capture prove with a poly-T sequence. Following the hybridization of the nucleic acid molecule 15 and the capture probe 12, a polymerase (or a reverse transcriptase in the case of RNA) is used to generate a first complimentary strand 23. For analysis of RNA, the first complimentary strand may be a cDNA strand. The first strand 23 comprises a compliment to nucleic acid molecule and the barcode sequence 201. The nucleic acid molecule 15-first complimentary strand 23 hybrid may be denatured (not shown) using any method traditional in the art, such as an exposure to a denaturing temperature.

FIG. 14 shows the complex of FIG. 13 , in which a second strand primer 24 comprising a random hexamer sequence anneals with the first strand 23 to form a DNA-primer hybrid. A DNA polymerase is used to synthesize a second complementary strand 25 complimentary to the first strand. The second complimentary strand comprises the sequence of the released nucleic acid molecule and the barcode unique to the droplet. Upon being denatured from the first complimentary strand, the second complimentary strand may be sequenced and the sequence of the barcode may be used to identify the droplet and cell from which the nucleic acid molecule was released. The barcoded nucleic acid molecules may then be used to accurately measure cellular response to the candidate compound.

Methods employing one or more of ligation tagging and capture probe tagging of nucleic acid molecules or proteins with droplet specific barcodes and/or UMIs may be performed.

The complement of a nucleic acid when aligned need not be perfect; stable duplexes may contain mismatched base pairs or unmatched bases. Those skilled in the art of nucleic acid technology can determine duplex stability empirically considering a number of variables including, for example, the length of the oligonucleotide, percent concentration of cytosine and guanine bases in the oligonucleotide, ionic strength, and incidence of mismatched base pairs.

FIG. 15 and FIG. 16 show a schematic representation of a method for rupturing the monodispersed droplets 10 according to aspects of this disclosure. The monodispersed droplets 10 are depicted as circles present in a fluid in the bottom portion of a test tube. The fluid comprising the encapsulations is topped with reagents such a high salt buffer (middle layer), and breaker reagent (top layer). The high salt buffer may comprise beta mercaptoethanol and/or DTT, or other reducing reagents. The breaker reagent may comprise perfluorooctanol (PFO). Incubation of the fluid with the high salt buffer and breaker reagent is preferably done on ice. Candidate compounds 11 may be provided to the monodispersed droplets 10 that are designed, together with the droplets, to be provided to the single cells within each droplet. For example, candidate compounds 11 may be provided to each monodispersed droplet 10 via a syringe or via diffusion. Alternatively, candidate compounds 11 may be encapsulated within a droplet designed to join with one of the monodispersed droplets 10, thereby providing the candidate compound to the monodispersed droplet.

FIG. 16 shows the monodispersed droplets of FIG. 15 following the steps of mixing by, for example, vortex, shearing 18, and/or spin 19. The monodispersed droplets 10 are broken, and two layers 20, 21, aqueous and oil are formed. Depending on the type of oil used, the oil layer may be the bottom layer or the top layer. The template particles and cells associated with them are present in the aqueous layer. In certain embodiments, candidate compounds 11 may then be presented to the cells and cellular response to the candidate compounds measured.

Nucleic acid molecules, including capture probe bound nucleic acid molecules, released nucleic acid molecules, or amplified nucleic acid molecules, may be attached to streptavidin-coated magnetic beads. For example, streptavidin-coated magnetic beads bound to biotin-labeled oligonucleotides comprising a bait sequence may be used. The bait sequence may be complementary to a primer sequence of the nucleic acid molecule, which may be for example, the one or more UMIs the droplet unique barcode on the released nucleic acid molecule. The streptavidin-coated magnetic beads comprising the bait sequence may then be incubated with the nucleic acid molecule to allow hybridization of complementary sequences. The nucleic acid molecule may first be incubated with biotin-labeled oligonucleotides comprising a bait sequence, wherein the bait sequence is complementary to one or more barcodes of the nucleic acid molecule to allow hybridization of complementary sequences. Following the incubation, streptavidin-coated magnetic beads are added and further incubated with the nucleic acid molecule/biotin-labeled oligonucleotide mixture to allow streptavidin-biotin binding. Incubation steps may be done on ice.

Alternatively, general nucleic acid capture beads may be used, for example polystyrene beads surrounded by a layer of magnetite and/or carboxyl molecules, such as beads with a similar surface characteristic to SPRI beads. SPRI beads may be as described in Deangelis et al. (1995) “Solid-phase reversible immobilization for the isolation of PCR products”, Nucleic Acids Res. 23(22):4742-3, incorporated by reference.

Proteins and other markers of cellular response may also be separated from mixtures and/or measured by any known methods.

Template particles used in the present invention may further comprise a capture moiety. The capture moiety acts to capture specific cells, for example, specific types of cells. The capture moiety may comprise an Acrylate-terminated hydrocarbon linker with biotin termination. The capture moiety may be attached to a target-specific capture element, for example aptamers and/or antibodies. Examples of capture moieties and methods thereof are disclosed in PCT application no. PCT/US2019/053426, incorporated herein by reference.

As described above, tubes may be selected based on the volume of sample from which cells need to be separated and/or based on the number of cells to be separated. For example, the tube may be single large tube, such as a conical centrifuge tube, such as a Falcon® as sold by Corning Inc., Corning, N.Y., for example a tube with a volume of less than 40 mL. Such tubes may be advantageous where the number of cells to be analyzed is between 100,000 and 1 million cells or greater than 1 million cells. This method is useful when screening candidate compounds against heterogeneous cell types and exploring complex cellular response pathways in complex tissues.

The tubes may also be wells, such as standard 96 sample well kit. The well may be part of a microplate with multiple wells each used a tube. The microplate may comprise any number of wells as desired, for example 6-1536 wells. Advantageously, the microplate may comprise 96 wells. Wells may be advantageous where the number of cells to be analyzed is about 100 tagged cells. This method is useful deep screening of candidate compounds against homogenous cells. For example, screening various candidate compounds against a single cell line once the cell line has been isolated, for example by methods of the present invention.

The tubes may also be centrifuge, microcentrifuge, or PCR tubes, such as those sold be Eppendorf ®, Hamburg, Germany. Such tubes, for example, may be between 0.1 and 6 mL and are advantageous where the number of cells to be analyzed is about 10,000 cells. This method is also useful when screening candidate compounds against heterogeneous cell types, and can be advantageous where samples are limited.

As described above, because cells are encapsulated in mono-dispersed droplets simultaneously, methods of the present invention are easily scaled for the analysis of any number of cells. For example, tubes may be selected to analyze at least 1 million cells, at least 2 million cells, at least 10 million, at least than 100 million cells, or 200 million cells of greater. Additionally, because cells are encapsulated simultaneously, for any tubes and any number of cells sample preparation for sequencing may be completed within one day, and can be completed within three hours. Moreover, preparation of samples within each tube may be completed in as little time as about 5 minutes or about 2 minutes.

Primers and/or reagents may be added to the monodisperse droplets after formation of the monodisperse droplets in the tube. Primers and/or reagents may be added in one step, or in more than one step. For instance, the primers may be added in two or more steps, three or more steps, four or more steps, or five or more steps. Regardless of whether the primers are added in one step or in more than one step, they may be added after the addition of a lysing agent, prior to the addition of a lysing agent, or concomitantly with the addition of a lysing agent. When added before or after the addition of a lysing agent, PCR primers may be added in a separate step from the addition of a lysing agent.

Incorporation by Reference 

1. A method for drug screening, the method comprising: combining template particles with a plurality of single cells in a tube; generating in the tube a plurality of monodispersed droplets simultaneously that encapsulate a single one of the template particles and single one of the single cells; providing to the single cells one or more candidate compounds; and measuring a cellular response to the one or more candidate compounds.
 2. The method of claim 1, wherein the one or more candidate compounds are provided to a droplet by the single template particle encapsulated by the droplet.
 3. The method of claim 2, wherein the one or more candidate compounds are provided to the droplet from the surface of the template particle.
 4. The method of claim 2, wherein the template particles comprise one or more compartments and one or more candidate compounds are provided to the cells from a compartment of the template particle.
 5. The method of claim 1, wherein the one or more candidate compounds are provided to the single cells after generating the plurality of monodispersed droplets.
 6. The method of claim 5, comprising the step of releasing the single cells from the monodispersed droplets prior to providing to the single cells one or more candidate drug compounds.
 7. The method of claim 1, wherein combining template particles and generating droplets comprises: combining the template particles with the single cells in a first fluid; adding a second fluid to the first fluid; and shearing the fluids to generate a plurality of monodispersed droplets simultaneously that contain a single one of the template particles and a single one of the single cells.
 8. The method of claim 7, wherein the first fluid and the second fluid are immiscible.
 9. The method of claim 7, wherein the first fluid comprises an aqueous phase fluid.
 10. The method of claim 7, wherein the second fluid comprises an oil.
 11. The method of claim 7, wherein shearing the fluids comprises vortexing, shaking, flicking, stirring, or pipetting.
 12. The method of claim 1, wherein the method further comprises releasing nucleic acid molecules from the single cells and sequencing the nucleic acid molecules.
 13. The method of claim 12, wherein releasing nucleic acid molecules from the single cells comprises lysing each of the single cells contained within the monodisperse droplets to release the nucleic acid molecules.
 14. The method of claim 13, wherein measuring a cellular response comprises sequencing the nucleic acid molecules.
 15. The method of claim 14, wherein the nucleic acid molecules are mRNA molecules.
 16. The method of claim 15, wherein measuring a cellular response comprises quantifying a plurality of distinct mRNA molecules in the cells associated with the drug response.
 17. The method of claim 16, wherein measuring a cellular response comprises generating a gene expression profile for each of the single cells.
 18. The method of claim 1, wherein measuring a cellular response comprises quantifying a plurality of protein molecules in the cells associated with a drug response.
 19. The method of claim 18, wherein measuring a cellular response comprises generating a protein expression profile for each of the single cells.
 20. The method of claim 1, wherein the tube is a conical centrifuge tube. 21-28. (canceled) 